Regulation of recombinant cardiac cystic fibrosis transmembrane conductance regulator chloride channels by protein kinase C

We investigated the regulation of cardiac cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels by protein kinase C (PKC) in Xenopus oocytes injected with cRNA encoding the cardiac (exon 5-) CFTR Cl- channel isoform. Membrane currents were recorded using a two-electrode voltage clamp technique. Activators of PKC or a cAMP cocktail elicited robust time-independent Cl- currents in cardiac CFTR-injected oocytes, but not in control water-injected oocytes. The effects of costimulation of both pathways were additive; however, maximum protein kinase A (PKA) activation occluded further activation by PKC. In oocytes expressing either the cardiac (exon 5-) or epithelial (exon 5+) CFTR isoform, Cl- currents activated by PKA were sustained, whereas PKC-activated currents were transient, with initial activation followed by slow current decay in the continued presence of phorbol esters, the latter effect likely due to down-regulation of endogenous PKC activity. The specific PKA inhibitor, adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS), and various protein phosphatase inhibitors were used to determine whether the stimulatory effects of PKC are dependent upon the PKA phosphorylation state of cardiac CFTR channels. Intraoocyte injection of 1,2-bis(2-aminophenoxy)ethane-N,N, N,N-tetraacetic acid (BAPTA) or pretreatment of oocytes with BAPTA-acetoxymethyl-ester (BAPTA-AM) nearly completely prevented dephosphorylation of CFTR currents activated by cAMP, an effect consistent with inhibition of protein phosphatase 2C (PP2C) by chelation of intracellular Mg2+. PKC-induced stimulation of CFTR channels was prevented by inhibition of basal endogenous PKA activity, and phorbol esters failed to stimulate CFTR channels trapped into either the partially PKA phosphorylated (P1) or the fully PKA phosphorylated (P1P2) channel states. Site-directed mutagenesis of serines (S686 and S790) within two consensus PKC phosphorylation sites on the cardiac CFTR regulatory domain attentuated, but did not eliminate, the stimulatory effects of phorbol esters on mutant CFTR channels. The effects of PKC on cardiac CFTR Cl- channels are consistent with a simple model in which PKC phosphorylation of the R domain facilitates PKA-induced transitions from dephosphorylated (D) to partially (P1) phosphorylated and fully (P1P2) phosphorylated channel states.     

Protein kinase C modulates inactivation of Kv3.3 channels

Modulation of some Kv3 family potassium channels by protein kinase C (PKC) regulates their amplitude and kinetics and adjusts firing patterns of auditory neurons in response to stimulation. Nevertheless, little is known about the modulation of Kv3.3, a channel that is widely expressed throughout the nervous system and is the dominant Kv3 family member in auditory brainstem. We have cloned the cDNA for the Kv3.3 channel from mouse brain and have expressed it in a mammalian cell line and in Xenopus oocytes to characterize its biophysical properties and modulation by PKC. Kv3.3 currents activate at positive voltages and undergo inactivation with time constants of 150-250 ms. Activators of PKC increased current amplitude and removed inactivation of Kv3.3 currents, and a specific PKC pseudosubstrate inhibitor peptide prevented the effects of the activators. Elimination of the first 78 amino acids of the N terminus of Kv3.3 produced noninactivating currents suggesting that PKC modulates N-type inactivation, potentially by phosphorylation of sites in this region. To identify potential phosphorylation sites, we investigated the response of channels in which serines in this N-terminal domain were subjected to mutagenesis. Our results suggest that serines at positions 3 and 9 are potential PKC phosphorylation sites. Computer simulations of model neurons suggest that phosphorylation of Kv3.3 by PKC may allow neurons to maintain action potential height during stimulation at high frequencies, and may therefore contribute to stimulus-induced changes in the intrinsic excitability of neurons such as those of the auditory brainstem.     

Modulation of cardiac sodium channel gating by protein kinase A can be altered by disease-linked mutation

Mutations associated with sodium channel-linked inherited Long-QT syndrome often result in a gain of channel function by disrupting channel inactivation. A small fraction of channels fail to inactivate (burst) at depolarized potentials where normal (wild type) channels fully inactivate. These noninactivating channels give rise to a sustained macroscopic current. We studied the effects of protein kinase A stimulation on sustained current in wild type and three disease-linked C-terminal mutant channels (D1790G, Y1795C, and Y1795H). We show that protein kinase A stimulation differentially affects gating in the mutant channels. Wild type, Y1795C, and Y1795H channels are insensitive to protein kinase A stimulation, whereas "bursting" in the D1790G mutant is markedly enhanced by protein kinase A-dependent phosphorylation. Our results suggest that the charge at position 1790 of the C terminus of the channel modulates the response of the cardiac sodium channel to protein kinase A stimulation and that phosphorylation of residue 36 in the N terminus and residue 525 in the cytoplasmic linker joining domains I and II of the channel alpha subunit facilitate destabilization of inactivation and thereby increase sustained current.     

Stimulation of the ERK pathway by GTP-loaded Rap1 requires the concomitant activation of Ras,protein kinase C,and protein kinase A in neuronal cells

The small GTPases Ras or Rap1 were suggested to mediate the stimulatory effect of some G protein-coupled receptors on ERK activity in neuronal cells. Accordingly, we reported here that pituitary adenylate cyclase-activating polypeptide (PACAP), whose G protein-coupled receptor triggers neuronal differentiation of the PC12 cell line via ERK1/2 activation, transiently activated Ras and induced the sustained GTP loading of Rap1. Ras mediated peak stimulation of ERK by PACAP, whereas Rap1 was necessary for the sustained activation phase. However, PACAP-induced GTP-loading of Rap1 was not sufficient to account for ERK activation by PACAP because 1) PACAP-elicited Rap1 GTP-loading depended only on phospholipase C, whereas maximal stimulation of ERK by PACAP also required the activity of protein kinase A (PKA), protein kinase C (PKC), and calcium-dependent signaling; and 2) constitutively active mutants of Rap1, Rap1A-V12, and Rap1B-V12 only minimally stimulated the ERK pathway compared with Ras-V12. The effect of Rap1A-V12 was dramatically potentiated by the concurrent activation of PKC, the cAMP pathway, and Ras, and this potentiation was blocked by dominant-negative mutants of Ras and Raf. Thus, this set of data indicated that GPCR-elicited GTP loading of Rap1 was not sufficient to stimulate efficiently ERK in PC12 cells and required the permissive co-stimulation of PKA, PKC, or Ras.     

Sequential phosphorylation mediates receptor- and kinase-induced inhibition of TREK-1 background potassium channels

Background potassium channels determine membrane potential and input resistance and serve as prominent effectors for modulatory regulation of cellular excitability. TREK-1 is a two-pore domain background K+ channel (KCNK2, K2P2.1) that is sensitive to a variety of physicochemical and humoral factors. In this work, we used a recombinant expression system to show that activation of G alpha(q)-coupled receptors leads to inhibition of TREK-1 channels via protein kinase C (PKC), and we identified a critical phosphorylation site in a key regulatory domain that mediates inhibition of the channel. In HEK 293 cells co-expressing TREK-1 and either the thyrotropin-releasing hormone receptor (TRHR1) or the Orexin receptor (Orx1R), agonist stimulation induced robust channel inhibition that was suppressed by a bisindolylmaleimide PKC inhibitor but not by a protein kinase A blocker ((R(p))-cAMP-S). Channel inhibition by agonists or by direct activators of PKC (phorbol dibutyrate) and PKA (forskolin) was disrupted not only by alanine or aspartate mutations at an identified PKA site (Ser-333) in the C terminus, but also at a more proximal regulatory site in the cytoplasmic C terminus (Ser-300); S333A and S300A mutations enhanced basal TREK-1 current, whereas S333D and S300D substitutions mimicked phosphorylation and strongly diminished currents. When studied in combination, TREK-1 current density was enhanced in S300A/S333D but reduced in S300D/S333A mutant channels. Channel mutants were expressed and appropriately targeted to cell membranes. Together, these data support a sequential phosphorylation model in which receptor-induced kinase activation drives modification at Ser-333 that enables subsequent phosphorylation at Ser-300 to inhibit TREK-1 channel activity.     

Calcium influx through L-type channels generates protein kinase M to induce burst firing of dopamine cells in the rat ventral tegmental area

Enhanced activity of the dopaminergic system originating in the ventral tegmental area is implicated in addictive and psychiatric disorders. Burst firing increases dopamine levels at the synapse to signal novelty and salience. We have previously reported a calcium-dependent burst firing of dopamine cells mediated by L-type channels following cholinergic stimulation; this paper describes a cellular mechanism resulting in burst firing following L-type channel activation. Calcium influx through L-type channels following FPL 64176 or (S)-(-)-Bay K8644 induced burst firing independent of dopamine, glutamate, or calcium from the internal stores. Burst firing induced as such was completely blocked by the substrate site protein kinase C (PKC) inhibitor chelerythrine but not by the diacylglycerol site inhibitor calphostin C. Western blotting analysis showed that FPL 64176 and (S)-(-)-Bay K8644 increased the cleavage of PKC to generate protein kinase M (PKM) and the specific calpain inhibitor MDL28170 blocked this increase. Prevention of PKM production by inhibiting calpain or depleting PKC blocked burst firing induction whereas direct loading of purified PKM into cells induced burst firing. Activation of the N-methyl-D-aspartic acid type glutamate or cholinergic receptors known to induce burst firing increased PKM expression. These results indicate that calcium influx through L-type channels activates a calcium-dependent protease that cleaves PKC to generate constitutively active and labile PKM resulting in burst firing of dopamine cells, a pathway that is involved in glutamatergic or cholinergic modulation of the central dopamine system.     

Identification of an integration center for cross-talk between protein kinase C and G protein modulation of N-type calcium channels

The modulation of presynaptic calcium channel activity by second messengers provides a fine tuning mechanism for neurotransmitter release. In neurons, the activation of certain G protein-coupled receptors reduces N-type channel activity by approximately 60%. In contrast, activation of protein kinase C (PKC) results in an approximately 50% increase in N-type channel activity, and subsequent G protein inhibition is antagonized. Here, we describe the molecular determinants that control the dual effects of PKC-dependent phosphorylation. The double substitution of two adjacent PKC consensus sites in the calcium channel domain I-II linker (Thr422, Ser425) to alanines abolished both PKC-dependent up-regulation and the PKC-G protein cross-talk. The single substitution of Ser425 to glutamic acid abolished PKC up-regulation but had no effect on G protein modulation. Replacement of Thr422 with glutamic acid eliminated PKC-dependent up-regulation and mimicked the effects of PKC phosphorylation on G protein inhibition. Our data suggest that Thr422 mediates the antagonistic effect of PKC on G protein modulation, while phosphorylation of either Thr422 or Ser425 are sufficient to increase N-type channel activity. Thus, Thr422 serves as a molecular switch by which PKC is able to simultaneously trigger the up-regulation of channel activity and antagonize G protein inhibition.     

Modulation of Ca-activated K channels of human erythrocytes by endogenous protein kinase C

Single IK_Ca channels of human erythrocytes were studied with the patch-clamp technique to define their modulation by endogenous protein kinase C (PKC). The perfusion of the cytoplasmic side of freshly excised patches with the PKC activator, phorbol 12-myristate 13-acetate (PMA), inhibited channel activity. This effect was blocked by PKC_19-31, a peptide inhibitor specific for PKC. Similar results were obtained by perfusing the membrane patches with the structurally unrelated PKC activator 1-oleoyl-2-acetylglycerol (OAG). Blocking of this effect was induced by perfusion with PKC_19-31 or chelerythrine. Channel activity was not inhibited by the PMA analog 4α-phorbol 12,13-didecanoate (4αPDD), which has no effect on PKC. Activation of endogenous cAMP-dependent protein kinase (PKA), which is known to up-modulate IK_Ca channels, restored channel activity previously inhibited by OAG. The application of OAG induced a reversible reduction of channel activity previously up-modulated by the activation of PKA, indicating that the effects of the two kinases are commutative, and antagonistic. Kinetic analysis showed that down-regulation by PKC mainly changes the opening frequency without significantly affecting mean channel open time and conductance. These results provide evidence that an endogenous PKC down-modulates the activity of native IK_Ca channels of human erythrocytes. Our results show that PKA and PKC signal transduction pathways integrate their effects, determining the open probability of the IK_Ca channels.     

Cyclic AMP-dependent protein kinase decreases GABAA receptor current in mouse spinal neurons

GABA, the major inhibitory neurotransmitter in the mammalian brain, binds to GABAA receptors, which form chloride ion channels. The predicted structure of the GABAA receptor places a consensus phosphorylation site for cAMP-dependent protein kinase (PKA) on an intracellular domain of the channel. Phosphorylation by various protein kinases has been shown to alter the activity of certain ligand- and voltage-gated ion channels. We have examined the role of phosphorylation by the catalytic subunit of PKA in the regulation of GABAA receptor channel function using whole-cell and excised outside-out patch-clamp techniques. Inclusion of the catalytic subunit of PKA in the recording pipettes significantly reduced GABA-evoked whole-cell and single-channel chloride currents. Both heat inactivation of PKA and addition of the specific protein kinase inhibitor peptide prevented the reduction of GABA-evoked currents by PKA. Neither mean channel open time nor channel conductance was affected by PKA. The reduction in GABA receptor current by PKA was primarily due to a reduction in channel opening frequency.     

Phosphorylation of S1505 in the cardiac Na+ channel inactivation gate is required for modulation by protein kinase C

Inactivation of both brain and cardiac Na+ channels is modulated by activation of protein kinase C (PKC) but in different ways. Previous experiments had shown that phosphorylation of serine 1506 in the highly conserved loop connecting homologous domains III and IV (LIII/IV) of the brain Na+ channel alpha subunit is necessary for all effects of PKC. Here we examine the importance of the analogous serine for the different modulation of the rH1 cardiac Na+ channel. Serine 1505 of rH1 was mutated to alanine to prevent its phosphorylation, and the resulting mutant channel was expressed in 1610 cells. Electrophysiological properties of these mutant channels were indistinguishable from those of wild-type (WT) rH1 channels. Activation of PKC with 1-oleoyl-2-acetyl-sn-glycerol (OAG) reduced WT Na+ current by 49.3 +/- 4.2% (P < 0.01) but S1505A mutant current was reduced by only 8.5 +/- 5.4% (P = 0.29) when the holding potential was -94 mV. PKC activation also caused a -17-mV shift in the voltage dependence of steady-state inactivation of the WT channel which was abolished in the mutant. Thus, phosphorylation of serine 1505 is required for both the negative shift in the inactivation curve and the reduction in Na+ current by PKC. Phosphorylation of S1505/1506 has common and divergent effects in brain and cardiac Na+ channels. In both brain and cardiac Na+ channels, phosphorylation of this site by PKC is required for reduction of peak Na+ current. However, phosphorylation of S1506 in brain Na+ channels slows and destabilizes inactivation of the open channel. Phosphorylation of S1505 in cardiac, but not S1506 in brain, Na+ channels causes a negative shift in the inactivation curve, indicating that it stabilizes inactivation from closed states. Since LIII/IV containing S1505/S1506 is completely conserved, interaction of the phosphorylated serine with other regions of the channel must differ in the two channel types.     

Dihydropyridine-sensitive skeletal muscle Ca channels in polarized planar bilayers. 3. Effects of phosphorylation by protein kinase C.

The effects of protein kinase C (PKC) were studied on dihydropyridine (DHP)-sensitive Ca channels from rabbit skeletal muscle T tubule membranes. To determine which channel subunits become phosphorylated under the conditions used for electrophysiological studies, we first performed biochemical studies of phosphorylation. T tubular membranes were fused with vesicles of the lipid mixture used in the planar bilayers, and phosphorylation was assessed using the same concentrations of PKC, adenosine 5''-triphosphate, and buffers as were used in the electrophysiological experiments. The alpha 1 subunit of the DHP receptors was phosphorylated by PKC to an extent of 1 mol phosphate/mol protein. The beta subunit was also phosphorylated but to a significantly lesser extent. The DHP-sensitive Ca channel activity was studied after fusing T tubule membranes with planar bilayers (Ma, J., C. Mundiña-Weilenmann, M. M. Hosey, and E. Ríos. 1991. Biophys. J. 60:890-901). The bilayers were held at -80 mV and activated by depolarizing voltage clamp pulses. The observed Ca channels exhibited two open states (tau o1 = 5 ms and tau o2 = 25 ms). On addition of purified PKC to the intracellular side, the proportion of the longer open state increased threefold. The average open probability during a 2-s, maximally activating pulse (Pmax) increased from 10 to 15%. The voltage dependence of activation was not changed by PKC; the Boltzmann parameters were V1 = -20.5 mV and K = 10.5 mV, which were not significantly different from the reference channels. The deactivation (closing) time constant was increased from 7 to 12 ms after PKC. The inactivation time constant during the pulse was slightly increased(from 1.2 to 1.6 s), and the channel availability at the holding potential was decreased from 76 to 71%. Taken together, the results revealed that PKC increased Pmax largely through a shift in the voltage independent open-close equilibrium of the fully activated channels.This is in contrast with the effect of phosphorylation by PKA (Mundir''a-Weilenmann, C., J. Ma, E. Rios, and M. M. Hosey. 1991. Biophys.J. 60:902-909), which also increases Pmax but mostly by increasing the availability of channels and slowing inactivation during the pulse.     

Activation of protein kinase C alters the intracellular distribution and mobility of cardiac Na+ channels

Na(+) current derived from expression of the cardiac isoform SCN5A is reduced by receptor-mediated or direct activation of protein kinase C (PKC). Previous work has suggested a possible role for loss of Na(+) channels at the plasma membrane in this effect, but the results are controversial. In this study, we tested the hypothesis that PKC activation acutely modulates the intracellular distribution of SCN5A channels and that this effect can be visualized in living cells. In human embryonic kidney cells that stably expressed SCN5A with green fluorescent protein (GFP) fused to the channel COOH-terminus (SCN5A-GFP), Na(+) currents were suppressed by an exposure to PKC activation. Using confocal microscopy, colocalization of SCN5A-GFP channels with the plasma membrane under control and stimulated conditions was quantified. A separate population of SCN5A channels containing an extracellular epitope was immunolabeled to permit temporally stable labeling of the plasma membrane. Our results demonstrated that Na(+) channels were preferentially trafficked away from the plasma membrane by PKC activation, with a major contribution by Ca(2+)-sensitive or conventional PKC isoforms, whereas stimulation of protein kinase A (PKA) had the opposite effect. Removal of the conserved PKC site Ser(1503) or exposure to the NADPH oxidase inhibitor apocynin eliminated the PKC-mediated effect to alter channel trafficking, indicating that both channel phosphorylation and ROS were required. Experiments using fluorescence recovery after photobleaching demonstrated that both PKC and PKA also modified channel mobility in a manner consistent with the dynamics of channel distribution. These results demonstrate that the activation of protein kinases can acutely regulate the intracellular distribution and molecular mobility of cardiac Na(+) channels in living cells.     

Regulation of MAP kinase activity by peptide receptor signalling pathway: paradigms of multiplicity

G protein-coupled receptors (GPCRs) can stimulate the mitogen-activated protein kinase (MAPK) cascade and thereby induce cellular proliferation like receptor tyrosine kinases (RTKs). Work over the past 5 years has established several models which reduce the links of G(i)-, G(q)-, and G(s)-coupled receptors to MAPK on few principle pathways. They include (i) Ras-dependent activation of MAPK via transactivation of RTKs such as the epidermal growth factor receptor (EGFR), (ii) Ras-independent MAPK activation via protein kinase C (PKC) that converges with the RTK signalling at the level of Raf, and (iii) activation as well as inactivation of MAPK via the cAMP/protein kinase A (PKA) pathway in dependency on the type of Raf. Most of these generalizing hypotheses are founded on experimental data obtained from expression studies and using a limited set of individual receptors. This review will compare these models with pathways to MAPK found for a great variety of peptide hormone and neuropeptide receptor subtypes in various cells. It becomes evident that under endogenous conditions, the transactivation pathway is less dominant as postulated, whereas pathways involving isoforms of PKC and, especially, phosphoinositide 3-kinase (PI-3K) appear to play a more important role as assumed so far. Highly cell-specific and unusual connections of signalling proteins towards MAPK, in particular tumour cells, might provide points of attacks for new therapeutic concepts.     

Transmitter modulation of slow,activity-dependent alterations in sodium channel availability endows neurons with a novel form of cellular plasticity

Voltage-gated Na+ channels are major targets of G protein-coupled receptor (GPCR)-initiated signaling cascades. These cascades act principally through protein kinase-mediated phosphorylation of the channel alpha subunit. Phosphorylation reduces Na+ channel availability in most instances without producing major alterations of fast channel gating. The nature of this change in availability is poorly understood. The results described here show that both GPCR- and protein kinase-dependent reductions in Na+ channel availability are mediated by a slow, voltage-dependent process with striking similarity to slow inactivation, an intrinsic gating mechanism of Na+ channels. This process is strictly associated with neuronal activity and develops over seconds, endowing neurons with a novel form of cellular plasticity shaping synaptic integration, dendritic electrogenesis, and repetitive discharge.     

Modulation of Ca2+-activated K+ channels of human erythrocytes by endogenous protein kinase C

Single IK(Ca) channels of human erythrocytes were studied with the patch-clamp technique to define their modulation by endogenous protein kinase C (PKC). The perfusion of the cytoplasmic side of freshly excised patches with the PKC activator, phorbol 12-myristate 13-acetate (PMA), inhibited channel activity. This effect was blocked by PKC(19-31), a peptide inhibitor specific for PKC. Similar results were obtained by perfusing the membrane patches with the structurally unrelated PKC activator 1-oleoyl-2-acetylglycerol (OAG). Blocking of this effect was induced by perfusion with PKC(19-31) or chelerythrine. Channel activity was not inhibited by the PMA analog 4alpha-phorbol 12,13-didecanoate (4alphaPDD), which has no effect on PKC. Activation of endogenous cAMP-dependent protein kinase (PKA), which is known to up-modulate IK(Ca) channels, restored channel activity previously inhibited by OAG. The application of OAG induced a reversible reduction of channel activity previously up-modulated by the activation of PKA, indicating that the effects of the two kinases are commutative, and antagonistic. Kinetic analysis showed that down-regulation by PKC mainly changes the opening frequency without significantly affecting mean channel open time and conductance. These results provide evidence that an endogenous PKC down-modulates the activity of native IK(Ca) channels of human erythrocytes. Our results show that PKA and PKC signal transduction pathways integrate their effects, determining the open probability of the IK(Ca) channels.     

Dual regulation of the n type K+ channel in Jurkat T lymphocytes by protein kinases A and C.

We have measured the activity of the n type K+ channel present in human (Jurkat) T lymphocytes using the patch clamp technique in the whole-cell configuration. We report that protein kinase A (PKA) and protein kinase C (PKC) modulate, in a dual manner, the K+ conductance in these cells. Activation of PKA decreases the amplitude of the current, as previously reported (Bastin, B., Payet, M. D., and Dupuis, G. (1990) Cell. Immunol. 128, 385-399), and this is also the case for 12-O-tetradecanoylphorbol-13-acetate-dependent activation of PKC. In contrast, inhibitors of PKC (H7, staurosporine, polymixin B, and anti-PKC antibody) increase the current amplitude. Of importance, down-regulation of PKC or its inhibition prevented the PKA-dependent inhibition of the K+ channels. Addition of alkaline phosphatase via the patch pipette increased the K+ conductance under basal conditions and reversed the inhibition produced by PKA. The dual modulation of K+ channels in Jurkat T cells is in agreement with the presence of consensus sequences in the primary structure of the n type K+ channel.     

Dual regulation of renal Kir7.1 potassium channels by protein Kinase A and protein Kinase C

The renal inward rectifier potassium channel Kir7.1 has been proposed to be functionally important for tubular K⁺ recycling and secretion. This study investigated the regulation of Kir7.1 by PKA and PKC.Cloned human Kir7.1 channels were expressed heterologously in Xenopus oocytes. After pharmacological PKC activation, Kir7.1 currents were strongly inhibited. Co-application of PKC inhibitors attenuated this effect. Inactivation of PKC consensus sites also strongly attenuated the effect with a single site (²⁰¹S) being essential for almost the total PKC sensitivity. In contrast, PKA activation induced an increase of Kir7.1 currents. This effect was absent in mutant Kir7.1 channels lacking PKA consensus site ²⁸⁷S.In summary, this study demonstrates the dual regulation of Kir7.1 channel function by PKA and PKC. Structurally, these regulations depend on two key residues in the C-terminal channel domain (^Ser201 for PKC and ^Ser287 for PKA).     

Rapid protein kinase D translocation in response to G protein-coupled receptor activation. Dependence on protein kinase C.

Protein kinase D (PKD)/protein kinase C (PKC) mu is a serine/threonine protein kinase that can be activated by physiological stimuli like growth factors, antigen-receptor engagement and G protein-coupled receptor (GPCR) agonists via a phosphorylation-dependent mechanism that requires PKC activity. In order to investigate the dynamic mechanisms associated with GPCR signaling, the intracellular translocation of a green fluorescent protein-tagged PKD was analyzed by real-time visualization in fibroblasts and epithelial cells stimulated with bombesin, a GPCR agonist. We found that bombesin induced a rapidly reversible plasma membrane translocation of green fluorescent protein-tagged PKD, an event that can be divided into two distinct mechanistic steps. The first step, which is exclusively mediated by the cysteine-rich domain in the N terminus of PKD, involved its translocation from the cytosol to the plasma membrane. The second step, i.e. the rapid reverse translocation of PKD from the plasma membrane to the cytosol, required its catalytic domain and surprisingly PKC activity. These findings provide evidence for a novel mechanism by which PKC coordinates the translocation and activation of PKD in response to bombesin-induced GPCR activation.     

A single Gbeta subunit locus controls cross-talk between protein kinase C and G protein regulation of N-type calcium channels

The modulation of N-type calcium channels is a key factor in the control of neurotransmitter release. Whereas N-type channels are inhibited by Gbetagamma subunits in a G protein beta-isoform-dependent manner, channel activity is typically stimulated by activation of protein kinase C (PKC). In addition, there is cross-talk among these pathways, such that PKC-dependent phosphorylation of the Gbetagamma target site on the N-type channel antagonizes subsequent G protein inhibition, albeit only for Gbeta(1)-mediated responses. The molecular mechanisms that control this G protein beta subunit subtype-specific regulation have not been described. Here, we show that G protein inhibition of N-type calcium channels is critically dependent on two separate but adjacent approximately 20-amino acid regions of the Gbeta subunit, plus a highly conserved Asn-Tyr-Val motif. These regions are distinct from those implicated previously in Gbetagamma signaling to other effectors such as G protein-coupled inward rectifier potassium channels, phospholipase beta(2), and adenylyl cyclase, thus raising the possibility that the specificity for G protein signaling to calcium channels might rely on unique G protein structural determinants. In addition, we identify a highly specific locus on the Gbeta(1) subunit that serves as a molecular detector of PKC-dependent phosphorylation of the G protein target site on the N-type channel alpha(1) subunit, thus providing for a molecular basis for G protein-PKC cross-talk. Overall, our results significantly advance our understanding of the molecular details underlying the integration of G protein and PKC signaling pathways at the level of the N-type calcium channel alpha(1) subunit.     

Coordination of membrane excitability through a GIRK1 signaling complex in the atria

Control of heart rate is a complex process that integrates the function of multiple G protein-coupled receptors and ion channels. Among them, the G protein-regulated inwardly rectifying K+ (GIRK or KACh) channels of sinoatrial node and atria play a major role in beat-to-beat regulation of the heart rate. The atrial KACh channels are heterotetrameric proteins that consist of two pore-forming subunits, GIRK1 and GIRK4. Following m2-muscarinic acetylcholine receptor (M2R) stimulation, KACh channel activation is conferred by the direct binding of G protein betagamma subunits (Gbetagamma) to the channel. Here we show that atrial KACh channels are assembled in a signaling complex with Gbetagamma, G protein-coupled receptor kinase, cyclic adenosine monophosphate-dependent protein kinase, two protein phosphatases, PP1 and PP2A, receptor for activated C kinase 1, and actin. This complex would enable the KACh channels to rapidly integrate beta-adrenergic and M2R signaling in the membrane, and it provides insight into general principles governing spatial integration of different transduction pathways. Furthermore, the same complex might recruit protein kinase C (PKC) to the KACh channel following alpha-adrenergic receptor stimulation. Our electro-physiological recordings from single atrial KACh channels revealed a potent inhibition of Gbetagamma-induced channel activity by PKC, thus validating the physiological significance of the observed complex as interconnecting site where signaling molecules congregate to execute a coordinated control of membrane excitability.